Wnt and frizzled receptors as targets for immunotherapy in head and neck squamous cell carcinomas

ABSTRACT

The diverse receptor-ligand pairs of the Wnt and frizzled (Fzd) families play important roles during embryonic development, and thus may be overexpressed in cancers that arise from immature cells. The mRNA levels and expression levels of 5 Wnt (Wnt-1, 5a, 7a, 10b, 13) and 2 Fzd (Fzd-2, 5) genes in 10 head and neck squamous carcinoma cell lines (HNSCC) were investigated. In addition, anti-Wnt-1 antibodies were used to study the Wnt/Fzd signalling pathway. These results indicate that HNSCC cell lines overexpress one or more Wnt and Fzd genes, and the growth and survival of a subset of HNSCC may depend on the Wnt/Fzd pathway. Therefore, The Wnt and Fzd receptors may be useful targets for immunotherapy of this common cancer.

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This patent application claims the benefit of the filing date ofU.S. Provisional Application No. 60/287,995, filed 1 May 2001 which isincorporated herein by reference.

STATEMENT OF RIGHTS TO INVENTIONS MADE UNDER FEDERALLY SPONSOREDRESEARCH

[0002] This invention was made with U.S. Government support under GrantAR 44850 awarded by the National Institutes of Health. The Governmentmay have certain rights in this invention.

TECHNICAL FIELD

[0003] This application relates to proteins involved in the Wnt/frizzledsignaling pathway. More specifically, it involves the role of theseproteins in proliferative disorders.

BACKGROUND OF THE INVENTION

[0004] Many cancers arise from differentiated tissues that are slowlydividing. The initial malignant population may have developed from asmall, rapidly proliferating population of residual tissue stem cells orcells with a less differentiated subcellular profile. A strategy fortargeting tumor cells that are antigenically distinct from maturedifferentiated cells could be useful in the treatment of cancer,particularly for controlling microscopic spread of disease. Malignantcells may express receptors used in embryonic patterning, which mayserve as immunologic targets distinct from mature differentiated tissue.

[0005] In embryogenesis body patterning is related to the axialexpression of different proteins. The proximal-distal axis is controlledby fibroblast growth factor (Vogel, A. et al., “Involvement of FGF-8 ininitiation, outgrowth and patterning of the vertebrate limb,”Development 122:1737-1750 (1996); Vogel, A. and Tickle, C., “FGF-4maintains polarizing activity of posterior limb bud cells in vivo and invitro,” Development 119:199-206 (1993); Niswander, L. et al., “FGF-4replaces the apical ectodermal ridge and directs outgrowth andpatterning of the limb,” Cell 75:579-587 (1993)), anterior-posterioraxis by Sonic hedgehog (Riddle, R. D. et al., “Sonic hedgehog mediatesthe polarizing activity of the ZPA,” Cell 75:1401-1416 (1993)), and thedorsal ventral axis by wingless (Parr, B. A. et al., “Mouse Wnt genesexhibit discrete domains of expression in the early embryonic CNS andlimb buds,” Development 119:247-261 (1993); Riddle, R. D. et al.,“Induction of the LIM homeobox gene Lmx1 by Wnt7a establishesdorsoventral pattern in the vertebrate limb,” Cell 83:631-640 (1995);Vogel, A. et al, “Dorsal cell fate specified by chick Lmx1 duringvertebrate limb development,” Nature 378:716-720 (1995)). These factorsare closely cross-regulated in development. The secretion of Wnt(wingless) is stimulated by Sonic hedgehog (SHH) signaling andconversely the expression of SHH is supported by the continued presenceof wingless. SHH in turn influences fibroblast growth factor (FGF)expression (Niswander, L. et al., “A positive feedback loop coordinatesgrowth and patterning in the vertebrate limb,” Nature 371:609-612(1994); Niswander, L., et al. “Function of FGF-4 in limb development,”Mol Reprod Dev 39:83-88; discussion 88-89 (1994); Laufer, E. et al.,“Sonic hedgehog and Fgf-4 act through a signaling cascade and feedbackloop to integrate growth and patterning of the developing limb bud,”Cell 79:993-1003 (1994)). Wingless is a ligand for a G-coupled proteinreceptor named frizzled, which mediates a complex signaling cascade(Vinson, C. R. and Adler, P. N., “Directional non-cell autonomy and thetransmission of polarity information by the frizzled gene ofDrosophila,” Nature 329:549-551 (1987)). Transcriptional regulation isalso mediated by SHH cell surface interaction with its ligand, Patched.Patched tonically inhibits signaling through Smoothened until it bindsto SHH. These pathways are illustrated in FIG. 1, which has been adaptedfrom reviews by others (Hunter, T., “Oncoprotein networks,” Cell88:333-346 (1997); Ng, J. K. et al., “Molecular and cellular basis ofpattern formation during vertebrate limb development,” Curr Top Dev Biol41:37-66 (1999); Ramsdell, A. F. and Yost, H. J., “Molecular mechanismsof vertebrate left-right development,” Trends Genet 14:459-465 (1998)).

[0006] Head and neck squamous cell carcinoma (HNSCC) is the sixth mostcommon cancer in developed countries, and of the 44,000 annual casesreported in the United States approximately 11,000 will result in anunfavorable outcome (Landis, S. H. et al., “Cancer statistics,” CACancer J Clin. 49, 8-31 (1999); Parkin, D. M. et al., “Global cancerstatistics,” CA Cancer J Clin. 49, 33-64 (1999)). Although metastaticHNSCC can respond to chemotherapy and radiotherapy, it is seldomadequately controlled. Therefore, it is important to identify newmolecular determinants on HNSCC that may be potential targets forchemotherapy or immunotherapy.

[0007] In APC-deficient colon carcinoma, beta-catenin accumulates and isconstitutively complexed with nuclear Tcf-4 (Sparks, A. B. et al.,“Mutational analysis of the APC/beta-catenin/Tcf pathway in colorectalcancer,” Cancer Res 58:1130-1134 (1998)). Other colon carcinomas andmelanomas also contain constitutive nuclear Tcf-4/beta-catenin complexesas a result of mutations in the N terminus of beta-catenin that renderit insensitive to downregulation by APC, and GSK3 beta (Morin, P. J. etal., “Activation of beta-catenin-Tcf signaling in colon cancer bymutations in beta-catenin or APC,” Science 275:1787-1790 (1997);Rubinfeld, B. et al. “Stabilization of beta-catenin by genetic defectsin melanoma cell lines,” Science 275:1790-1792 (1997)). This results inthe unregulated expression of Tcf-4 oncogenic target genes, such asc-myc, cyclin D1, and c-jun (He, T. C. et al., “Identification of c-MYCas a target of the APC pathway,” Science 281:1509-1512 (1998); Shtutman,M. et al, “The cyclin D1 gene is a target of the beta-catenin/LEF-1pathway,” Proc. Nat'l. Acad. Sci. USA 96:5522-5527 (1999); Li, L. etal., “Disheveled proteins lead to two signaling pathwvays. Regulation ofLEF-1 and c-Jun N-terminal kinase in mammalian cells,” J Biol Chem274:1129-134 (1999)). The expression of covalently linked beta-cateninand LEF-1 has been directly demonstrated to result in the oncogenictransformation of chicken fibroblasts (Aoki, M. et al., “Nuclearendpoint of Wnt signaling: neoplastic transformation induced bytransactivating lymphoid-enhancing factor 1,” Proc. Nat'l. Acad. Sci.USA 96:139-144 (1999)). Similar mechanisms leading to deregulation ofTcf target gene activity are likely to be involved in melanoma (Rimm, D.L. et al., “Frequent nuclear/cytoplasmic localization of beta-cateninwithout exon 3 mutations in malignant melanoma,” Am J Pathol 154:325-329(1999)), breast cancer (Bui, T. D. et al., “A novel human Wnt gene,WNT10B, maps to 12q13 and is expressed in human breast carcinomas,”Oncogene 14:1249-1253 (1997)),heptocellular carcinoma (de La Coste, A.et al., “Somatic mutations of the beta-catenin gene are frequent inmouse and human heptocellular carcinomas,” Proc Nat'l. Acad. Sci. USA95:8847-8851 (1998)), ovarian cancer (Palacios, J., and Gamallo, C.,“Mutations in the beta-catenin gene (CTNNB1) in endometrioid ovariancarcinomas,” Cancer Res 58:1344-1347 (1998)), endometrial cancer (Ikeda,T., “Mutational analysis of the CTNNB1 (beta-catenin) gene in humanendometrial cancer: frequent mutations at codon 34 that cause nuclearaccumulation,” Oncol Rep. 7:323-326 (2000)), medulloblastoma (Hamilton,S. R. et al., “The molecular basis of Turcot's syndrome,” N. Engl J Med332:839-847 (1995)), pilomatricomas (Chan, E. F et al. “A common humanskin tumour is caused by activating mutations in beta-catenin,” Nat.Genet 21:410-413 (1999)), and prostate cancer (Iozzo, R. V. et al.,“Aberrant expression of the growth factor Wnt-5A in human malignancy,”Cancer Res 55:3495-3499 (1995)).

[0008] Other growth regulation pathways in tumors have also attractedrecent interest. Many epithelial tumors express excess amounts ofepidermal growth factor-receptor tyrosine kinases, particularlyepidermal growth factor receptor (EGFR, or ErbB-1), and HER2 (ErbB-2)(Coussens, L. et al., “Tyrosine kinase receptor with extensive homologyto EGF receptor shares chromosomal location with neu oncogene,” Science230:1132-1139 (1985); King, C. R. et al., “Amplification of a novelv-erbB-related gene in a human mammary carcinoma,” Science 229:974-976(1985)). HER2 is transmembrane tyrosine kinase receptor, which dimerizeswith another member of the EGFR family to form an active dimericreceptor (Akiyama, T. et al., “The product of the human c-erbB-2 gene: a185-kilodalton glycoprotein with tyrosine kinase activity,” Science232:1644-1646 (1986)). The resulting phosphorylation of tyrosineresidues initiates complex signaling pathways that ultimately lead tocell division. HER2 is overexpressed in 25 to 30 percent of breastcancers, usually as a result of gene amplification (Slamon, D. J. etal., “Studies of the HER-2/neu proto-oncogene in human breast andovarian cancer,” Science 244:707-712 (1989)). High levels of thisprotein is associated with an adverse prognosis (Slamon, D. J. et al.,“Human breast cancer: correlation of relapse and survival withamplification of the HER-2/neu oncogene,” Science 235:177-182 (1987);Ravdin, P. M. and Chamness, G. C., “The c-erbB-2 proto-oncogene as aprognostic and predictive marker in breast cancer: a paradigm for thedevelopment of other macromolecular markers—a review,” Gene 159:19-27(1995)).

[0009] In the past decade there has been tremendous progress inidentifying genetic and molecular changes that occur duringtransformation of malignant cells. Many malignant cells have a lessdifferentiated phenotype, and a higher growth fraction than normal inadult tissues. These basic characteristics are similar to immature orembryonic cells. During the development of the embryo, various cellsurface receptors and ligands direct tissue pattern formation, andcellular differentiation (Hunter, T., “Oncoprotein networks,” Cell 88,333-346 (1997); Ng, J. K. et al., “Molecular and cellular basis ofpattern formation during vertebrate limb development,” Curr Top DevBiol. 41, 37-66 (1999); Ramsdell, A. F. and Yost, H. J., “Molecularmechanisms of vertebrate left-right development,” Trends Genet. 14,459-465 (1998)). The expression of these receptors and ligands is oftenno longer required in fully matured adult tissues. Because they areexpressed on the cell surface, the receptors and ligands important formorphologic patterning and tissue differentiation could be targets forthe immunotherapy of tumors that have arisen from residual immaturecells, or that have undergone de-differentiation.

[0010] Genes of the wingless (Wnt) and frizzled (Fzd) class have anestablished role in cell morphogenesis and cellular differentiation(Parr, B. A. et al., “Mouse Wnt genes exhibit discrete domains ofexpression in the early embryonic CNS and limb buds,” Development, 119,247-261 (1993); Riddle, R. D. et al., “Induction of the LIM homeoboxgene Lmx1 by WNT7a establishes dorsoventral pattern in the vertebratelimb,” Cell 83, 631-640 (1995); Vogel, A. et al., (1995) “Dorsal cellfate specified by chick Lmx1 during vertebrate limb development,” Nature378, 716-720 (1995)). The Wnt proteins are extracellular ligands for theFzd receptors, which resemble typical G protein coupled receptors(GPCRs). The first member of the 19 known human Wnt genes, Wnt-1, wasinitially discovered because of its oncogenic properties (Nusse, R. andVarmus, H. E., “Many tumors induced by the mouse mammary tumor viruscontain a provirus integrated in the same region of the host genome,”Cell 31, 99-109 (1982)). The Wnt glycoproteins bind to one or more ofthe 9 known, 7 transmembrane domain G-protein coupled Fzd receptors, toinitiate a chain of signaling events that often culminates in thestabilization and nuclear translocation of β-catenin, with resultantheterodimerization with one of the four members of the LEF/TCF family oftranscription factors (Cadigan, K. M. and Nusse, R., “Wnt signaling: acommon theme in animal development,” Genes Dev., 11, 3286-3305 (1997);Miller, J. R. et al., “Mechanism and function of signal transduction bythe Wnt/β-catenin and Wnt/Ca2+ pathways,” Oncogene 18, 7860-7872(1999)). These transcription factor complexes control the activities ofspecific Wnt target genes, including developmental regulators and othergenes involved in coordinating cell proliferation, cell-cellinteractions, and cell-matrix interactions (Vogel, A. and Tickle, C.,“FGF-4 maintains polarizing activity of posterior limb bud cells in vivoand in vitro,” Development 119:199-206 (1993)). The overexpression ofβ-catenin and LEF-1 has been demonstrated to result in the oncogenictransformation of chicken fibroblasts (Aoki, M. et al., “Nuclearendpoint of Wnt signaling: neoplastic transformation induced bytransactivating lymphoid-enhancing factor 1,” Proc. Nat'l. Acad. Sci.USA 96, 139-144 (1999)).

[0011] A recent survey using microarray techniques showed that mostHNSCC overexpress mRNAs of the Wnt family (Leethanakul, C. et al.,“Distinct pattern of expression of differentiation and growth-relatedgenes in squamous cell carcinomas of the head and neck revealed by theuse of laser capture microdissection and cDNA arrays,” Oncogene 19,3220-3224 (2000)). However, the various Wnt mRNAs are very homologous,and hybridization in microarrays often cannot distinguish betweenclosely related templates.

[0012] A murine monoclonal antibody 4DS binds with high affinity to theextracellular domain of HER2, thereby blocking its function in signaltransduction (Hudziak, R. M. et al. “p185HER2 monoclonal antibody hasantiproliferative effects in vitro and sensitizes human breast tumorcells to tumor necrosis factor,” Mol Cell Biol 9:1165-1172 (1989);Fendly, B. M. et al. “Characterization of murine monoclonal antibodiesreactive to either the human epidermal growth factor receptor orHER2/neu gene product,” Cancer Res 50:1550-1558 (1990); Fendly, B. M. etal. “The extracellular domain of HER2/neu is a potential immunogen foractive specific immunotherapy of breast cancer,” J Biol Response Mod9:449-455 (1990)). In experimental models of breast cancer, it wasactive in vitro and in vivo, and had greater anti-tumor effects whencombined with chemotherapy Hudziak, R. M. et al. “p185HER2 monoclonalantibody has antiproliferative effects in vitro and sensitizes humanbreast tumor cells to tumor necrosis factor,” Mol Cell Biol 9:1165-1172(1989); Pietras, R. J. et al., “Antibody to HER-2/neu receptor blocksDNA repair after cisplatin in human breast and ovarian cancer cells,”Oncogene 9:1829-1838 (1994). A recently completed phase 3 randomizedclinical trial of a humanized form of 4DS monoclonal antibody,trastuzumab (Herceptin; Genentech, Inc, South San Francisco, Calif.),demonstrated efficacy against some forms of breast tumors overexpressingHER2 (Slamon, D. J. et al., “Use of chemotherapy plus a monoclonalantibody against HER2 for metastatic breast cancer that overexpressesHER2,” N Engl J Med 344:783-792 (2001).

SUMMARY OF THE INVENTION

[0013] A method for determining overexpression of a wnt or frizzled genein a tumor cell comprising:

[0014] (a) isolating messenger RNA from a tumor cell and from acorresponding normal cell from the same source;

[0015] (b) performing reverse transcription PCR on the tumor cell andthe normal cell utilizing primers directed against regions of the wnt orfrizzled gene that are non-homologous with a known wnt gene or a knownfrizzled gene and obtaining amplicons from the reverse transcriptionPCR;

[0016] (c) assessing the concentrations of the resulting amplicons ofstep (b) in comparison to a control housekeeping gene; and

[0017] (d) identifying tumor cells that overexpress a wnt or frizzledgene at about five fold compared to normal cells tissue from the samesource

[0018] An isolated antibody directed against at least one sequence thatcorresponds to a non-homologous regions of a known wnt gene or a knownfrizzled gene.

[0019] A method of detecting overexpression of at least one wnt and/orfrizzled protein in a cancer cell compared to a non-cancer cellcomprising:

[0020] (a) contacting the cancer cell with an antibody directed againstat least one sequence that corresponds to a non-homologous region of aknown wnt or a known frizzled gene;

[0021] (b) contacting the non-cancer cell with the same antibody as instep (a);

[0022] (c) comparing the interaction of the antibody with the cancercell to the interaction of the antibody with the non-cancer cell; and

[0023] (d) correlating the interactions in (c) with the expressionlevels of the wnt and/or frizzled protein in both the cancer cell andthe non-cancer cell.

[0024] A method for altering the growth of a cell overexpressing atleast one wnt and/or frizzled protein comprising:

[0025] contacting the cell with a non-crossreactive antibody against thewnt and/or frizzled protein.

[0026] A method for altering the growth of a cell overexpressing atleast one wnt and/or frizzled protein comprising:

[0027] contacting the cell with a synthetic peptide, a recombinantprotein,

[0028] or a DNA vector, or any combination of a synthetic peptide and arecombinant protein and a DNA vector, comprising at least onenon-homologous region of known wnt and/or frizzled proteins.

BRIEF DESCRIPTION OF THE DRAWINGS

[0029]FIG. 1. Several developmental signaling pathways are depicted.

[0030]FIG. 2. RT-PCR analysis of a subset of HNSCC and B-cell lines forfrizzled 2 mRNA.

[0031]FIG. 3. A western blot analysis of tumor and normal cells forfrizzled 2, wnt1 and 10b.

[0032]FIGS. 4A, 4B, and 4C. An inhibition of proliferation assay in aHNSCC line is depicted. Specifically, anti-frizzled 2, anti-wnt 1, andanti-wnt 10b are tested for their ability to inhibit proliferation.

[0033]FIG. 5. Apoptotic effects of inhibition of the Wnt/Frizzledsignaling pathway in a HNSCC line is depicted.

[0034]FIG. 6. Sequence alignment of a portion of the first extracellularregion of human Frizzled receptors is depicted.

[0035]FIGS. 7A and 7B. FIG. 7A depicts an immunoblot after treatmentwith Wnt 1 or Wnt 10b antibodies. SNU1076 cells were treated for 72 hrswith 2 μg/ml of anti-Wnt 1, Wnt 10b, or control antibodies. FIG. 7Bshows that treatment with Wnt1 antibodies reduces transcription ofTCF/LEF gene.

[0036]FIGS. 8A and 8B. FIG. 8A depicts an RT-PCR amplification forWnt/FZD families in cancer cell lines. FIG. 8B depicts an RT-PCRamplification for Wnt/FZD families in normal cells.

[0037]FIGS. 9A and 9B. Protein expression of FZD 2, Wnt 1, Wnt 10b,β-catenin and actin in normal and malignant cells.

[0038]FIG. 10. Inhibition of proliferation of the SNU 1076 cell line Wnt1 and Wnt 10b.

[0039]FIG. 11. Growth inhibition with a soluble WNT antagonist, secretedfrizzled related protein (SFRP).

[0040]FIG. 12. Apoptotic effect of inhibition of the Wnt/Frizzledsignaling pathway in a HNSCC line.

DETAILED DESCRIPTION OF THE DRAWINGS

[0041]FIG. 1. Schematic of developmental signaling pathways is depicted.The signalling pathways of the Wnt/wingless and Hedehog/Sonic hedgehogare shown. Both sets of ligands interact with a cell surface receptor.Proteins involved in the signalling pathway are shown, for example, LEF1and GSK3.

[0042]FIG. 2. RT-PCR analysis of a subset of HNSCC and B-cell lines forfrizzled 2 mRNA. Total RNA was extracted from HNSCC lines (PCI13,Detroit 562, RPMI 2650, SNU1076, KB, AMC4), a CLL line (Lesch), aBurkitt lymphoma line (Ramos), glioma lines (U87MG, and U373MG), normalhuman bronchial epithelial cell lines (Clonetics, San Diego, Calif.) andnormal oral squamous epithelial (OSE) cells using RNAzol (Gibco BRL,Grand Island, N.Y.). Reverse transcription was performed using 1 μg ofRNA from each sample and the Superscript™ Preamplification kit (GibcoBRL). Frizzled 2 was amplified with 25 cycles of PCR. G3PDH mRNA wasamplified in a separate reaction for each sample.

[0043]FIG. 3. A sample western blot analysis of tumor and normal cellsfor frizzled 2, wnt 5A and 10b. Adherent cells in culture were harvestedand lysed with a solution containing 25 mM Tris HCl, 150 mM KCl, 5 mMEDTA, 1% NP-40, 0.5% sodium deoxycholic acid, 0.1% sodium dodecylsulfate, 1 mM NaVO₃, 1 mM NaF, 20 mM β-glycerophosphate and proteaseinhibitors. Twenty μg of protein from each cell line was separated bySDS-PAGE and transferred to a PVDF membrane. The membrane was immersedin 2% I-block, 0.05% Tween X in PBS and then incubated with a 1:500dilution of polyclonal goat anti-human Wnt 1, Wnt 10b, or frizzled 2 IgG(Santa Cruz Biotechnology, Santa Cruz, Calif.). These primary antibodieswere then detected by horseradish peroxidase-conjugated donkey anti-goatIgG (Santa Cruz) and chemiluminescence (ECL detection reagents, AmershamLife Science, Aylesbury, UK). To verify relative amount of proteintransferred in each lane, presence of actin was measured with an actinmonoclonal antibody (Chemi-Con International Inc, Temecula, Calif.).

[0044]FIGS. 4A, 4B, and 4C. Inhibition of proliferation in a HNSCC line.Briefly, either 7.5×10³ or 10×10³ SNU1076 cells per well were seeded in96 well plates. After 24 hours, graded amounts of polyclonal goatanti-human frizzled 2, Wnt 1, or Wnt 10b IgG (sAB)(Santa CruzBiotechnology, Santa Cruz, Calif.), or control goat anti-human IgG(cAB)(Fisher Scientific, Pittsburgh, Pa.) were added. On days 1, 2, 3,or 4, 20 μl of MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide)-based solution was added to wells for four hoursprior to lysis with 15% SDS, 0.015 M HCl. Absorbencies of 570 and 650 nmwere measured.

[0045]FIG. 5. Apoptotic effect of inhibition of the Wnt/Frizzledsignaling pathway in a HNSCC line. The HNSCC line SNU1076, growing inRPMI-1640 supplemented with 10% FBS, was treated for 72 hrs with 300ng/ml anti-Frizzled 2, Wnt-1, Wnt10b, or control nonspecific polyclonalantibodies. The cytotoxic effects of these antibodies were assessed byvital dye retention and DNA content. Panel A: cells were detached fromthe flasks by trypsin treatment and incubated for 10 minutes in growingmedium with 5 μg/ml Propidium iodide (PI) and 40 nM DiOC₆ and analyzedby flow cytometry. Viable cells (stripes) had high DiOC₆ (FL-1) and lowPI (FL-3) fluorescence, and apoptotic cells (stippled) had low DiOC₆(FL-1) and low PI (FL-3) fluorescence. Panel B: cells were detached fromthe flasks by trypsin treatment and incubated overnight in a hypotonicbuffer (0.1% citrate, 0.1% SDS) containing 50 μg/ml PI and 100 μg/mlRNase. The amount of DNA was then measured by flow cytometry, andapoptotic cells were defined as having a DNA content lower than the G₀G₁levels (sub-G₀ cells).

[0046]FIG. 6. Sequence alignment of a portion of the first extracellularregion of human Frizzled receptors. Specifically, the amino acidsequences of HFZ1 through HFZ10 are aligned to show similarity.

[0047]FIGS. 7A and 7B. FIG. 7A: immunoblot after treatment with Wnt 1 orWnt 10b antibodies. SNU1076 cells were treated for 72 hrs with 2 μg/mlof anti-Wnt 1, Wnt 10b, or control antibodies. Twenty μg of protein fromeach cell line was separated by SDS-PAGE and transferred to a PVDFmembrane. The membrane was immersed in 2% I-block, 0.05% Tween X in PBSand then incubated with a monoclonal anti-human β-catenin, cyclin D1, orfibronectin IgG. These primary antibodies were then detected byhorseradish peroxidase-conjugated anti-IgG and chemiluminescence. Toverify and compare relative amounts of protein in each lane, PVDFmembrane was stripped with Re-Blot™ Western blot recycling kit andreprobed for other antibodies or actin monoclonal antibody. FIG. 7B:treatment with Wnt1 antibodies reduces transcription of TCF/LEF gene.SNU 1076 cells were treated with 2 μg/ml of anti-Wnt-1, or controlantibodies for 36 hrs. SNU 1076 cells were cotransfected with 0.5 μg/mlof pTOPFLASH-Luc or pFOPFLASH-Luc and 0.5 μg/ml of pCMV-βGal. Cells wereharvested 24 h after transfection, and lysed in lysis buffer. Luciferaseand β-galactosidase activities determined using Dual-Light™ reportergene assay system. Luciferase activities of each of pTOPFLASH-Luc orpFOPFLASH-Luc and β-galactosidase activities of pCMV-βGal were measuredin the same sample by luminometer. Transfection efficiency of eachsample was normalized by the activity of β-galactosidase activity.

[0048]FIGS. 8A and 8B. FIG. 8A: RT-PCR amplification for Wnt/FZDfamilies in cancer cell lines. Lane 1: DNA standard, lane 2: H₂O, Lanes3 and 4: glioblastoma, lanes 5-14: head and neck cancers, lanes 15 and16: B cell cancers. FIG. 8B: RT-PCR amplification for Wnt/FZD familiesin normal cells. Lane 1: DNA standard, lane 2: H₂O, lanes 7 and 14:normal human bronchial epithelial cell, other lanes: normal oral squmouscells.

[0049]FIGS. 9A and 9B. Protein expression of FZD 2, Wnt 1, Wnt 10b,β-catenin and actin in normal and malignant cells. Normal oral squamousepithelium (OSE), normal human broncheotracheal epithelial cells (NHBE),HNSCC lines, and other solid and B cell tumor lines were lysed,separated by SDS-page, blotted onto PDVF membranes and successivelyprobed with the indicated antibodies.

[0050]FIG. 10. Inhibition of proliferation of the SNU 1076 cell line.7.5×10³ SNU 1076 cells per well were seeded in 96 well plates. After 24hours, graded amounts of polyclonal goat anti-human Wnt 1, Wnt 10b, orcontrol goat anti-human IgG were added. On days 1, 2, 3, or 4, 20 μl ofMTT solution was added to wells for four hours prior to lysis with 15%SDS, 0.015 M HCl. Absorbencies of 570 and 650 nm were measured. Data areexpressed as the mean of at least 4 independent experiments±SD.

[0051]FIG. 11. Growth inhibition with a soluble WNT antagonist, secretedfrizzled related protein (SFRP). Cell viability of two HNSCC lines wasdetermined with MTT assay 72 hours after addition of 2 μg/ml ofrecombinant human SFRP 1. Data are expressed as the mean of 2independent experiments±SD.

[0052]FIG. 12. Apoptotic effect of inhibition of the Wnt/Frizzledsignaling pathway in a HNSCC line. SNU1076 was treated for 72 hrs with 2μg/ml of anti-Wnt 1, Wnt 10b, or control antibodies. The cytotoxiceffects of these antibodies were assessed by vital dye retention and DNAcontent. Cells were detached from the flasks by trypsin treatment andincubated for 10 minutes in growing medium with 5 μg/ml Propidium iodide(PI) and 40 nM DiOC₆ and analyzed by flow cytometry. Viable cells hadhigh DiOC₆ (FL-1) and low PI (FL-3) fluorescence, and apoptotic cellshad low DiOC₆ (FL-1) and low PI (FL-3) fluorescence.

DETAILED DESCRIPTION OF THE INVENTION

[0053] Different clonal populations of HNSCC overexpress variousreceptors of the Wnt and Fzd family because of their immature cell oforigin and because of a growth and survival advantage provided byautocrine or paracrine Wnt/Fzd signaling. We examined HNSCC and normalhuman epithelial cell lines for the expression of 5 Wnt and 2 Fzd genes.The results showed that most HNSCCs did overexpress one or more Wnt andFzd mRNAs. Moreover, the Wnt/Fzd pathway was functional in some of theHNSCC cells, as indicated by the constitutive expression of a LEF/TCFreporter gene. In the SNU 1076 cell line, anti-Wnt-1 or anti-Wnt-10bantibodies decreased the expression of β-catenin and cyclin D1,inhibited cell growth, and induced apoptosis. Thus, the Wnt and Fzdgenes are frequently overexpressed in HNSCC, and are attractive targetsfor both immunotherapy and drug therapy.

[0054] We have examined tumor and normal cell lines for proteins thatare involved in embryonic development. These studies suggest that atleast one G-coupled protein receptor, frizzled 2, is overexpressed bymany tumor cell lines. A broader panel of normal and malignant cells canbe studied and immunization strategies can be developed directed towardspassive and active immunotherapies against this antigen.

[0055] Based on the successful experience of trastuzumab as anadjunctive passive immunotherapy as described above, an evaluation ofblocking the Wnt-frizzled signaling pathway on the growth of a HNSCCline with commercially available polyclonal antibodies was performed(FIGS. 4 and 5). Soluble inhibitors of fizzled have been described toinduce apoptosis secondary to their inhibition of frizzled signaling(Zhou, Z. J. et al., “Up-regulation of human secreted fizzled homolog inapoptosis and its down-regulation in breast tumors,” Int J Cancer78:95-99 (1998)). The antibodies tested appear to have slowed the growthof the tumor line and resulted in apoptosis (FIGS. 4 and 5).

[0056] To evaluate Wnt and Fzd receptors as potential tumor associatedantigens in head and neck squamous cell cancers (HNSCC), we screenedvarious tumor and normal cell lines by both RT-PCR, and immunoblotting.Initial screening revealed that both frizzled 2 and frizzled 5 areexpressed in head and neck squamous cell cancers (HNSCC), glioma, andchronic lymphocytic leukemia (CLL) (FIG. 2). Further, the resultsrevealed that Fzd-2 was overexpressed in many HNSCC cells, compared tonormal human bronchoepithelial (NHBE) cells (Table 1). The amino acidsequence of Fzd-2 is very homologous to Fzd-1 and 7 (Sagara, N. et al.“Molecular cloning, differential expression, and chromosomallocalization of human frizzled-1, frizzled-2, and frizzled-7,” BiochemBiophys Res Comm 252, 117-122 (1998)). To confirm that frizzled 2 wasspecifically amplified in the tumor

lines to RT-PCR products from selected reactions were cloned into the TAvector (Invitrogen, Carlsbad, Calif.) and sequenced. There was 100%identity of the inserts with the human frizzled 2 sequence by BLASTsearch. In addition, immunoblotting showed a lack of detectable Fzd-2protein in the lysates of NHBE in which there were weakly detectable orundetectable products by RT-PCR. The human Fzd-2 gene originally wasisolated by Sagara and colleagues (Sagara 1998, infra). Theseinvestigators also found that the mRNA for Fzd-2 was not detectable inany of 15 different normal human adult tissues, with the possibleexception of heart. In contrast, embryonic tissues, as well as six ofeight malignant cell lines, expressed abundant Fzd-2 mRNA. However,these investigators did not test for the expression of frizzled Fzd-2protein, and mRNA levels do not necessarily correlate with proteinexpression. Our studies show that Fzd-2 protein expression is prominentin HNSCC cell lines, when compared to normal NHBE cells. Hence,antibodies against specific determinants of the extracellular domain ofFzd-2 could be used to bind to and target such malignant cells.

[0057] Compared to NHBE cells, the HNSCC cell lines expressed muchhigher message levels of Wnt-1, Wnt-5a, Wnt-10b and Wnt-13. Of these Wntproteins Wnt-1, 5A, and 10b were exclusively expressed by the malignantcell lines and were not detected in the normal tissues tested.Immunoblotting experiments confirmed the overexpression of Wnt-1 andWnt-10b protein in several HNSCC cell lines (FIG. 3). Since the tumorshad high levels of both the ligands and their Fzd-2 receptors, it wasimportant to determine if Wnt/Fzd signaling was constitutively active inthe HNSCC cells. The canonical Wnt/Fzd signaling cascade leads to theaccumulation of cytoplasmic β-catenin and its translocation to thenucleus. In the nucleus beta-catenin binds a specific sequence motif atthe N terminus of lymphoid-enhancing factor/T cell factor (LEF/TCF) togenerate a transcriptionally active complex (Behrens J et al.“Functional interaction of beta-catenin with the transcription factorLEF-1,” Nature 382, 638-642 (1996)). Experiments using LEF/TCF reportergene, TOPFLASH, demonstrated that LEF/TCF dependent transcription wasactive in the SNU 1076 cells.

[0058] The Wnt/frizzled pathway has been previously implicated intumorigenesis. Soluble Wnt glycoproteins have been demonstrated totransmit signal by binding to the seven transmembrane domain G-proteincoupled-receptor frizzled (FIG. 1) (Bhanot, P. et al. “A new member ofthe frizzled family from Drosophila functions as a Wingless receptor,”Nature 382:225-230 (1996); Yang-Snyder, J. et al. “A frizzled homologfunctions in a vertebrate Wnt signaling pathway,” Curr Biol 6:1302-1306(1996); Leethanakul, C. et al. “Distinct pattern of expression ofdifferentiation and growth-related genes in squamous cell carcinomas ofthe head and neck revealed by the use of laser capture microdissectionand cDNA arrays,” Oncogene 19:3220-3224 (2000)). Upon Wnt signaling, acascade is initiated that results in the accumulation of cytoplasmicbeta-catenin and its translocation to the nucleus. In the nucleusbeta-catenin binds a specific sequence motif at the N terminus oflymphoid-enhancing factor/T cell factor (LEF/TCF) to generate atranscriptionally active complex (Behrens, J. et al. “Functionalinteraction of beta-catenin with the transcription factor LEF-1,” Nature382:638-642 (1996)). Beta-catenin interacts with multiple other proteinssuch as cadherin, which it links to the cytoskeleton (Hoschuetzky, H. etal. “Beta-catenin mediates the interaction of the cadherin-catenincomplex with epidermal growth factor receptor,” J Cell Biol127:1375-1380 (1994); Aberle, H. et al., “Assembly of thecadherin-catenin complex in vitro with recombinant proteins,” J Cell Sci107:3655-3663 (1994)). It also associates with the adenomatous polyposiscoli (APC) tumor suppressor protein and glycogen synthetase 3 beta(GSK3β) (Rubinfeld, B. et al., “Binding of GSK3beta to theAPC-beta-catenin complex and regulation of complex assembly,” Science272:1023-1026 (1996)). These proteins function to negatively regulatebeta catenin by facilitating phosphorylation near the aminoterminus andthus accelerating its proteolytic degradation (Yost, C. et al., “Theaxis-inducing activity, stability, and subcellular distribution ofbeta-catenin is regulated in Xenopus embryos by glycogen synthase kinase3,” Genes Dev 10:1443-1454 (1996)).

[0059] A panel of tumor cells that can be screened are derived from thepanel of 60 lines which are being characterized in the NationalInstitutes of Health Developmental Therapeutics Program. The cell linesthat are currently available include: (Non-Small Cell Lung Cancer)A549/ATCC, NCI-H226, NCI-H460, HOP-62, HOP-92, (colon cancer) HT29,HCT-116, (breast cancer) MCF7, NCI/ADR-RES, MDA-MB-231/ATCC, T-47D,(ovarian cancer) OVCAR-3, OVCAR-4, SK-OV-3, (leukemia) CCRF-CEM, K-562,MOLT-4, HL-60 (TB), RPMI-8226, (renal cell) 786-0, TK-10, (prostatecancer) PC-3, DU-145. Normal control cell lines can be purchased fromClonetics.

[0060] Although Wnt and Fzd were expressed in HNSCC cells, they may bedispensable for cell growth and survival. Therefore, the effects ofantibodies to the extracellular domains of Wnt-1 and Wnt-10b werestudied in three HNSCC lines known to express the receptors. Whencompared to control antibodies, both anti-Wnt antibodies slowed thegrowth of one of the HNSCC cell lines (SNU 1076) and resulted inapoptosis. Treatment with high levels of SFRP1, a Wnt antagonist,exerted a similar effect. Moreover, interference with Wnt/frizzledsignaling in SNU 1076 cells decreased the activity of the LEF/TCFreporter gene, and reduced levels of β-catenin cyclin D1 andfibronectin. These results suggest that continued autocrine or paracrineWnt/Fzd signaling may be required for the growth and survival of asubset of HNSCC cells.

[0061] These results suggest that antibodies against Wnt and frizzledreceptors may exert two different effects in HNSCC cancers in vivo. Inmalignant cells that depend on Wnt/Fzd signaling for survival, theantibodies might directly slow tumor growth and/or induce apoptosis. InHNSCC cells that incidentally overexpress the receptors, but do notrequire them for proliferation, the antibodies still could potentiallytarget the tumor cells for killing by complement, or antibody dependentcellular toxicity. Based on these data, we believe that passiveimmunotherapy could be a useful adjunctive therapy in HNSCC thatoverexpress one or more Wnt and Fzd receptors.

[0062] Experimental Methods

[0063] Cell lines and culture: Ten HNSCC, 2 B lymphoma, and 2glioblastoma cell lines were studied. Detroit-562 (pharyngeal cancer),KB (carcinoma in the floor of the mouth), RPMI-2650 (nasal septal)cancer), SCC-25 (tongue cancer), U87MG and U373MG (glioblastoma), Ramos(lymphoma), Detroit-551 (human skin fibroblast-like cells) and WI-38(human lung fibroblasts) were purchased from the American Type CultureCollection (Manassas, Va.). The PCI-1, 13, and 50 cell lines were kindlyprovided by Dr. T. Whiteside (Univ. of Pittsburgh, Pa.) (Whiteside, T.L. et al., “Human tumor antigen-specific T lymphocytes andinterleukin-2-activated natural killer cells: comparisons of antitumoreffects in vitro and in vivo,” Clin Cancer Res. 4, 1135-1145 (1998);Yasumura, S. et al., “Human cytotoxic T-cell lines with restrictedspecificity for squamous cell carcinoma of the head and neck,” CancerRes. 53, 1461-1468 (1993)). The HNSCC cell lines SNU 1066, SNU 1076 andAMC 4 cell lines were provided by Dr. J. G. Park (Seoul NationalUniversity, Korea) and Dr. S. Y. Kim (University of Ulsan, Korea),respectively (Ku, J. L. et al., “Establishment and characterization ofhuman laryngeal squamous cell carcinoma cell lines,” Laryngoscope 109,976-82 (1999); Kim, S. Y. et al. “Establishment and characterization ofnine new head and neck cancer cell lines,” Acta Otolaryngol. 117,775-784 (1997)). Two different normal human tracheobronchial epithelial(NHBE) cells derived from different persons were purchased fromClonetics (San Diego, Calif.). All cancer cell lines were cultured at37° C. in a humidified atmosphere of 5% CO₂, in either RPMI 1640, DMEM(Dulbecco's modified Eagle's medium), or Ham's12-DMEM medium, asrecommended by the suppliers, supplemented with 10% fetal bovine serum.NHBE cells were cultured in the bronchial epithelial cell growth mediaprovided by the company. Normal epithelial cells were obtained fromscrapings of the oral mucosa of 10 normal healthy volunteers. All celllines were found to be free of mycoplasma contamination.

[0064] RT-PCR Analyses: Total RNA was extracted by using Trizol® (GibcoBRL, Grand Island, N.Y.), according to the manufacturer's directions.Different pairs of gene-specific primers based on GenBank sequences ofcloned human Wnt and Fzd genes were used for reverse transcriptase-PCR(RT-PCR) analysis. Reverse transcription was performed with aSuperscript™ Preamplification kit (Gibco BRL). One microgram of RNA wasused from each sample, and 25-35 cycles of PCR were carried out. The PCRproducts were separated by electrophoresis, visualized under ultraviolet light, and scanned with a laser densitometer. The intensities ofthe Wnt and Fzd bands were compared with the amplicon of thehousekeeping gene G3PDH. Preliminary experiments confirmed that the PCRamplifications had not reached a plateau for all data reported in theresults. The following list summarizes the primer pairs used:

[0065] Fzd-2: 5′-cagcgtcttgcccgaccagatcca-3′(reverse);5′-ctagcgccgctcttcgtgtacctg-3′ (forward). Fzd-5:5′-ttcatgtgcctggtggtgggg-3′ (forward); 5′-tacacgtgcgacagggacacc-3′(reverse). Wnt-1: 5′-cacgacctcgtctacttcgac-3′ (forward);5′-acagacactcgtgcagtacgc-3′ (reverse). Wnt-5a:5′-acacctctttccaaacaggcc-3′ (forward); 5′-ggattgttaaactcaactctc-3′(reverse) Wnt-7a: 5′-cgcaacaagcggcccaccttc-3′ (forward),5′-tccgtgcgctcgctgcacgtg-3′ (reverse) Wnt-10b:5′-gaatgcgaatccacaacaacag-3′ (forward);5′-ttgcggttgtgggtatcaatgaa-3′(reverse). Wnt-13:5′-aagatggtgccaacttcaccg-3′ (forward);5′-ctgccttcttggggctttgc-3′(reverse) G3PDH: 5′-accacagtccatgccatcac -3′(forward); 5′-tacagcaacagggtggtgga-3′(reverse).

[0066] The specificities of the Wnt and Fzd PCR products were confirmedby cloning and sequencing the products, using a TOPO TA Cloning kit andM13 primers (Invitrogen, Carlsbad, Calif.).

[0067] Immunoblotting: After removal of medium, cells in logarithmicgrowth were disrupted in lysis buffer [25 mM Tris HCl, 150 mM KCl, 5 mMEDTA, 1% NP-40, 0.5% sodium deoxycholic acid, 0.1% sodium dodecylsulfate] including phosphatase and protease inhibitor cocktails. Eachlane of an SDS-PAGE gel was loaded with 20 μg of protein. Afterelectrophoresis, the proteins were transferred to a polyvinylidenedifluoride (PVDF) membrane, blocked with 2% I-block™ (Tropix Tnc.Bedford, Mass.) containing 0.05% Tween-X in PBS, and then incubated withprimary antibody. Horseradish peroxidase-conjugated anti-IgG (Santa CruzLaboratories, Santa Cruz, Calif.) was used as the secondary antibody.The membranes were developed using a chemiluminescence system (ECLdetection reagent: Amersham Life Science, Aylesbury, UK), and scannedwith a laser densitometer. The membranes were stripped with Re-Blot™Western blot recycling kit (Chemi-Con International Inc, Temecula,Calif.) and reprobed using other antibodies and actin monoclonalantibody (Chemi-Con International Inc) as a control. Prestainedmolecular weight markers (New England Biolabs, Beverly, Mass.) were usedas reference.

[0068] Antibodies: Polyclonal antibodies specific for the amino terminalextracellular domains of Wnt-1 and Wnt-10b, and for the carboxy terminalregion of Fzd-2, were purchased from Santa Cruz Laboratories, andmonoclonal antibodies specific for β-catenin and fibronectin werepurchased from Transduction Laboratories (Lexington, Ky.). Antibodies tocyclin D1 and actin were purchased from PharMingen (San Diego, Calif.)and Chemi-Con International Inc., respectively. Purified recombinanthuman soluble frizzled-related protein-1 was prepared in Dr. J. Rubin'slaboratory as described previously (Uren, A. et al., “Secretedfrizzled-related protein-1 binds directly to Wingless and is a biphasicmodulator of Wnt signaling,” J Biol Chem. 275, 4374-4382 (2000)).

[0069] MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazoliumbromide)-based cell assay: Cell proliferation was determined by acolorimetric MTT assay. Briefly, either 7.5−10×10³ cells were dispersedin each well of a 96 well plate. Twenty-hours after culture, 4 differentconcentrations of anti-Wnt-1 or ant-Wnt-10b antibody (2 μg/ml, 0.2μg/ml, 20 ng/ml, and 2 ng/ml) were added to the cultures. The sameconcentrations of goat antihuman IgG (Fisher Scientific) were used as anisotype control. The antibodies were dialyzed against tissue culturemedium prior to use, to remove preservatives. On 1, 2, 3, or 4 daysafter incubation, 20 μl of MTT solution was added to each well. Fourhours later the cells were lysed, and absorbances at 570 nM and 650 nMwere measured and growth, as a percentage of control, was determinedfrom the formula:

% of control growth=(B−A)/(C−A)×100

[0070] where A=absorbance at start of incubation, B=absorbance afterincubation with antibodies tested, C=absorbance after incubation withcontrol antibody. The assays were performed in triplicate, and theresults represent the mean value±standard deviation from fourindependent experiments.

[0071] Flow Cytometry: Cell apoptosis was assayed by propidium iodide(PI) and DIOC₆ staining, followed by flow cytometry. The HNSCC line,SNU1076, was treated with 2 μg/ml anti-Wnt-1, anti-Wnt-10, or controlIgG for 72 hrs. Cells were detached from the flasks by trypsin treatmentand incubated for 10 minutes in medium with 5 μg/ml PI and 40 nM DiOC₆,and then were analyzed by flow cytometry in a FACS caliber(Becton-Dickinson, San Jose, Calif.). Viable cells had high DiOC₆ (FL-1)and low PI (FL-3) fluorescence, whereas apoptotic cells had low DiOC₆(FL-1) and low PI (FL-3) fluorescence.

[0072] Tumor and normal cell lines can be identified that expressfrizzled 2. Ten cell lines that express frizzled 2 and at least two celllines that do not are currently being tested. The cells will be platedas described above for FIG. 4. The mouse sera that tests for highesttiter and specificity in aim 2 will be used in the cell cultures. Thecells will be exposed to graded amounts of polyclonal anti-frizzled 2mouse sera and normal control serum. On days 1, 2, 3, and 4 subsets ofthe replicate wells will be assayed for proliferative capacity. Onsuccessive days 20 μl of MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide)-based solution will be added to wells for fourhours prior to lysis with 15% SDS, 0.015 M HCl. Absorbencies of 570 and650 nm will measured. These measurements will be performed in triplicateand statistical relevance will be assessed by Students t test forP<0.05.

[0073] The selected cell lines will also undergo analysis for DNAcontent by Propidium iodide (PI) staining. Cell lines treated for 72hours in the presence of graded concentrations of normal or immunizedmouse serum will be trypsinized, incubated for 10 minutes with 5 μg/mlPI and 40 nM DiOC₆, and analyzed by flow cytometry. Viable cells will beDiOC₆ (FL-1) high and PI (FL-3) low, and apoptotic cells will be DiOC₆(FL-1) low and PI (FL-3) low. Additionally, cells will detached from theflasks with trypsin and incubated overnight in a hypotonic buffer (0.1%citrate, 0.1% SDS) containing 50 μg/ml PI and 100 μg/ml RNase. Theamount of DNA will be measured by flow cytometry. Apoptotic cells aredefined as having a DNA content lower than the G₀G₁ levels (sub-G₀cells).

[0074] Transient Luciferase Assays: The pTOPFLASH-Luc reporter genevector and the pFOPFLASH-Luc control were kindly provided by Dr. HansClevers (University Medical Center Utrecht, The Netherlands). ForTOPFLASH/FOPFLASH reporter gene assays, SNU 1076 cells werecotransfected with 0.5 μg of pTOPFLASH-Luc or pFOPFLASH-Luc and 0.5 μgof pCMV-βGal, as described previously (Korinek, V. et al., “Constitutivetranscriptional activation by a beta-catenin-Tcf complex in APC−/−coloncarcinoma,” Science 275, 1784-1787 (1997)). Cells were harvested 24 hafter transfection, disrupted in lysis buffer, and luciferase andβ-galactosidase activities were determined using the Dual-Light reportergene assay system (Applied Biosystems, Foster City, Calif.). Luciferaseactivities of each pTOPFLASH-Luc or pFOPFLASH-Luc transfected culture,and the β-galactosidase activities of pCMV-βGal transfected cells, weremeasured in the same samples using a luminometer. The transfectionefficiencies of the samples were normalized by the activity ofβ-galactosidase.

[0075] Other features and advantages of the invention will be apparentfrom the detailed description and from the claims.

[0076] The present invention is further described by the followingexamples. The examples are provided solely to illustrate the inventionby reference to specific embodiments. These exemplifications, whileillustrating certain specific aspects of the invention, do not portraythe limitations or circumscribe the scope of the disclosed invention.

EXAMPLES Example 1 Immunogenicity of Isolated Non-Homologous Regions ofFrizzled 2

[0077] The first extracellular domain of frizzled 2 contains a regionwhich based on protein structure is least homologous to the otherfrizzled protein family members (FIG. 6) (Sagara, N. et al. “Molecularcloning, differential expression, and chromosomal localization of humanfrizzled-1, frizzled-2, and frizzled-7,” Biochem Biophys Res Commun252:117-122 (1998)). This polypeptide sequence may have sufficientternary structure to generate an antibody response to the nativeprotein. In order to enhance B cell stimulation this epitope will becoupled to T cell epitopes that have been described to generate T cellhelp.

[0078] The overall strategy will be to use the least conserved region ofthe frizzled protein, attempting to preserve the most native structurepossible and to generate the most potent immune response. The mostversatile method for designing vaccines of defined regions is nakedplasmid DNA. The advantages are that the vectors can be rapidlyredesigned to change the length of sequence that is expressed,discontinuous regions of the protein can be co-expressed, and the DNAsequence of the protein can be fused to other epitopes to enhanceantigenicity (O'Hern, P. A. et al. “Colinear synthesis of anantigen-specific B-cell epitope with a ‘promiscuous’ tetanus toxinT-cell epitope: a synthetic peptide immunocontraceptive,” Vaccine15:1761-1766 (1997); Paterson, M. et al., “Design and evaluation of aZP3 peptide vaccine in a homologous primate model,” Mol Hum Reprod5:342-352 (1999); Dakappagari, N. K. et al., “Prevention of mammarytumors with a chimeric HER-2 B-cell epitope peptide vaccine,” Cancer Res60:3782-3789 (2000)). It affords the versatility of expressing soluble,membrane bound proteins, or small peptide fragments. Also gene transferby this technique is a powerful tool to introduce multiple proteinelements into the same or separate locations. In this system single ormultiple proteins can be locally expressed. Injecting a combination ofplasmids expressing antigens and costimulators like B7.1 and B7.2results in enhanced immune responses (Corr, M. et al., “Costimulationprovided by DNA immunization enhances antitumor immunity,” J Immunol159:4999-5004 (1997); Chan, K. et al., “The roles of mhc class ii, cd40,and b7 costimulation in ctl induction by plasmid dna (DNA?),” J Immunol166:3061-3066 (2001)).

[0079] Several plasmids have been constructed which are under thecontrol of the cytomegalovirus (CMV) promoter which has been found toenable high levels of antigen expression in injected muscle. The pCMVintvector includes the cytomegalovirus (CMV) E1 promoter, the simian virus(SV40) t-intron, and the SV-40 polyadenylation site (Corr, M. et al.“Gene vaccination with naked plasmid DNA: mechanism of CTL priming,” JExp Med 184:1555-1560 (1996)). The ACB vector has the same elementsexcept the polyadenylation sequence is from the bovine growth hormonegene (Sato, Y. et al. “Immunostimulatory DNA sequences necessary foreffective intradermal gene immunization,” Science 273:352-354 (1996)).The first set of plasmid constructs planned will encode the leasthomologous region of the frizzled 2 between the ninth and tenthcysteines. These cysteines will be preserved in this series ofconstructs as they may stabilize a configuration that enables antibodybinding to the native protein. This polypeptide fragment will be fusedat the aminoterminus or the carboxylterminus via a short linker to atetanus toxin or measles virus fusion (MVF) protein T helper epitopes(see below) (O'Hern, P. A. et al. “Colinear synthesis of anantigen-specific B-cell epitope with a ‘promiscuous’ tetanus toxinT-cell epitope: a synthetic peptide immunocontraceptive,” Vaccine15:1761-1766 (1997); Paterson, M. et al “Design and evaluation of a ZP3peptide vaccine in a homologous primate model,” Mol Hum Reprod 5:342-352(1999); Dakappagari, N. K. et al., “Prevention of mammary tumors with achimeric HER-2 B-cell epitope peptide vaccine,” Cancer Res 60:3782-3789(2000)). These minigenes will be constructed with overlappingoligonucleotides. The oligonucleotides are 5′ prime phosphorylated withT4 kinase at room temperature for 30 minutes, annealed by boiling anequimolar admixture of two complementary oligomers and slow cooling. Thedouble stranded oligonucleotides are then ligated 3′ to the tissueplasminogen leader (TPA) leader into the EcoR47III site in frame andinto the BamH1 site of the pBluescript SKII vector. The minigene is thensubcloned into the pCMV and pACB vectors between the Pst1 and Xba1 sitesas previously described (Corr, M. et al., “Costimulation provided by DNAimmunization enhances antitumor immunity,” J Immunol 159:4999-5004(1997)).

[0080] The inserts for the vectors are designed as described above. Thefrizzled putative B cell epitope is from the published sequence. Thetetanus toxin and measles MVF T helper epitopes have been optimized forhuman codon usage by the most frequently used codon per amino acid. TheDNA constructs have an initiating methionine and stop codons added tothe 5′ and 3′ ends respectively. The aminoacid and DNA sequences aresummarized below with the short GPSL linker sequence in bold and the Tcell helper epitope underlined.Tetanus toxin epitope fused to a frizzled domain pFZD2-TTMCVGQNHSEDGAPALLTTAPPPGLQPGAGGTPGGPGGGGAPPRYATLEHPFHC-GPSL-VDDALINSTKIYSYFPSV-STOP ATG TGC GTC GGC CAG AAC CAC TCC GAG GACGGA GCT CCC GCG CTA CTC ACC ACC GCG CCG CCG CCG GGA CTG CAG CCG GGT GCCGGG GGC ACC CCG GGT GGC CCG GGC GGC GGC GGC GCT CCC CCG CGC TAC GCC ACGCTG GAG CAC CCC TTC CAC TGC-GGC CCC AGC CTG-GTG GAC GAC GCC CTG ATC AAC AGC ACC AAG ATC TAC AGC TAC TTTCCC AGC GTG TAG pTT-FZD2 MVDDALINSTKIYSYFPSV-GPSL-CVGQNHSEDGAPALLTTAPPPGLQPGAGGTPGGPGGGGAPPRYATLEHPFHC-STOPATG GTG GAC GAC GCC CTG ATC AAC AGC ACC AAG ATC TAC AGC TAC TTT CCC AGCGTG-GGC CCC AGC CTG-TGC GTC GGC CAG AAC CAC TCC GAG GAC GGA GCT CCC GCGCTA CTC ACC ACC GCG CCG CCG CCG GGA CTG CAG CCG GGT GCC GGG GGC ACC CCGGGT GGC CCG GGC GGC GGC GGC GCT CCC CCG CGC TAC GCC ACG CTG GAG CAC CCCTTC CAC TGC TAG Measles MVF epitope fused to a frizzled domainPFZD2-MMVF MCVGQNHSEDGAPALLTTAPPPGLQPGAGGTPGGPGGGGAPPRYATLEHPFHC-GPSL-KLLSLIKGVIVHRLEGVE-STOP ATG TGC GTC GGC CAG AAC CAC TCC GAG GAC GGA GCTCCC GCG CTA CTC ACC ACC GCG CCG CCG CCG GGA CTG CAG CCG GGT GCC GGG GGCACC CCG GGT GGC CCG GGC GGC GGC GGC GCT CCC CCG CGC TAC GCC ACG CTG GAGCAC CCC TTC CAC TGC-GGC CCC AGC CTG-AAG CTG CTG AGC CTG ATC AAG GGC GTG ATC GTG CAC CGC CTG GAGGGC GTG GAG TAG PMMVF-FZD2 MKLLSLIKGVIVHRLEGVE-GPSL-CVGQNHSEDGAPALLTTAPPPGLQPGAGGTPGGPGGGGAPRYATLEHPFHC-STOPATG AAG CTG CTG AGC CTG ATC AAG GGC GTG ATC GTG CAC CGC CTG GAG GGC GTGGAG-GGC CCC AGC CTG-TGC GTC GGC CAG AAC CAC TCC GAG GAC GGA GCT CCC GCGCTA CTC ACC ACC GCG CCG CCG CCG GGA CTG CAG CCG GGT GCC GGG GGC ACC CCGGGT GGC CCG GGC GGC GGC GGC GCT CCC CCG CGC TAC GCC ACG CTG GAG CAC CCCTTC CAC TGC TAG

[0081] Plasmid DNA is prepared using Qiagen Maxiprep (Chatsworth,Calif.) kits with the modification of adding one tenth volume 10% TritonX-114 (Sigma, St. Louis, Mo.) to the clarified bacterial lysate prior toapplying it to a column. Prior to injection the residual endotoxin levelis quantified using a limulus extract clot assay (Associates of CapeCod, Woods Hole, Mass.). A level of ≦5 ng endotoxin/μg DNA need beobtained prior to use in an animal (Corr, M. et al. “In vivo priming byDNA injection occurs predominantly by antigen transfer,” J Immunol163:4721-4727 (1999)). The DNA is resuspended in a sterile pyrogen freesaline solution for injection.

[0082] Twenty-eight female mice will be divided into groups of 4 miceeach. They will be injected in the dermis of the tail with a combinationof 50 μg plasmid encoding a costimulator (B7-1 or B7-2) and 50 μg linkerplasmid diluted in normal saline at weeks zero, one and two. A groupwith empty vector is included as a negative control. The groups are asfollows: Group Plasmid 1 Plasmid 2 A pTT-FZD2 nCMV B pTT-FZD2 nCMVB7-1 CpTT-FZD2 nCMVB7-2 D pFZD2-TT nCMV E pFZD2-TT nCMVB7-1 F pFZD2-TTnCMVB7-2 G — nCMV

[0083] Another group of mice in similar groups will be immunized usingthe pMMVF-FZD2 and pFZD2-MMVF set of linked epitope plasmids. ThenCMVB7-1 and nCMVB7-2 constructs encode the cDNAs for murine CD80 andCD86, which were kindly provided by G. Freeman (Dana-Farber CancerInstitute, Boston, Mass.) (Corr, M. et al., “Costimulation provided byDNA immunization enhances antitumor immunity,” J Immunol 159:4999-5004(1997)).

[0084] Mice will be bled prior to the start of the experiment and thenevery two weeks thereafter. Serum will be separated and stored at −20°C. prior to testing. On week ten (seven weeks after the last injection)mice will be sacrificed. The titers of antibody will be tested byanti-peptide ELISA. Ninety-six well plates (Costar) are coated with 50μl/well 20 μg/ml peptide in phosphate buffered saline (PBS) overnight at4° C. The plates are then washed and blocked with 200 μl/well 2% bovineserum albumin (BSA) in PBS. Sera are diluted in 2% BSA in PBS. Afterovernight incubation at 4° C. the plates are washed. Bound murine IgG isdetected by alkaline phosphatase conjugated-goat anti-murine IgG(Jackson Immunoresearch Laboratories) followed by p-nitrophenylphosphatesubstrate. The titration curves for each sera are compared usingDeltaSOFT II v. 3.66 (Biometallics, Princeton, N.J.).

[0085] Mice that develop sufficiently high titers of antibody that bindto the peptide will be tested for specificity to frizzled 2 byfluorescent cytometry with cells that express the protein bytransfection and known tumor cells that have the mRNA. We will also testthe binding by Western blot analysis of cells that express this isoformand to cells that have been found to express other frizzled familymembers. Briefly, immunoblotting will be performed as described above.Cells are lysed in with a solution containing 25 mM Tris HCl, 150 mMKCl, 5 mM EDTA, 1% NP-40, 0.5% sodium deoxycholic acid, 0.1% sodiumdodecyl sulfate, 1 mM NaVO₃, 1 mM NaF, 20 mM β-glycerophosphate andprotease inhibitors. Twenty μg of protein from each cell line isseparated by SDS-PAGE and transferred to a PVDF membrane. The membraneis soaked in 2% I-block, 0.05% Tween X in PBS and then incubated with a1:500 dilution of polyclonal pre or post immunization mouse serum at1:500 dilution. Murine antibody binding is then detected by horseradishperoxidase-conjugated rat anti-mouse IgG and chemiluminescence (ECLdetection reagents). To verify relative amount of protein transferred ineach lane, the blots are then stripped and the presence of actin ismeasured with an actin monoclonal antibody.

[0086] Different immunization strategies are being evaluated for theirefficacy in eliciting a humoral immune response. If the antibodyresponse is weak then the vectors can be redesigned with other knownpotent T helper epitopes. Other vectors can be designed where thepolypeptide from frizzled 2 is shorter and does not contain thecysteines, which may be inhibiting the most desirable conformation.Another immunization strategy will be to use a prime boost method. Theanimals are originally injected with plasmid DNA and then are boostedwith peptide or recombinant protein in incomplete Freund's adjuvant. TheB-cell epitope in each construct may need to be redesigned until thereis no cross-reactivity in the humoral response to other frizzledisoforms.

Example 2 Expression of Wnt and Fzd mRNAs in HNSCC

[0087] Ten different HNSCC cell lines, two normal humanbroncho-epithelial (NHBE) cell lines, and normal oral squamousepithelial cells were tested by RT-PCR for the expression of five Wnts(Wnt-1, Wnt-5a, Wnt-7a, Wnt-10b, Wnt-13), and two Fzds (Fzd-2 and 5).Representative results are illustrated in FIG. 8 and are summarized inTable 1. When compared to the housekeeping gene G3PDH, all the Wnts, aswell as Fzd-2, were expressed more frequently in HNSCC than in normalcells, while there was no difference in Fzd-5 gene expression. Of theWnt genes, Wnt-1, 5a, and 10b were most strongly expressed by themalignant cells, but were barely detectable in the normal tissuestested. We then investigated further Wnt-1 and Wnt-10b, since these Wntssignal through the canonical β-catenin and LEF/TCF, and becauseantibodies to the extracellular domains were available.

Example 3 Expression of Wnt/Fzd Proteins in HNSCC

[0088] Cell lines were lysed and analyzed for Wnt-1, Wnt-10b, Fzd-2, andβ-catenin protein expression by immunoblotting (FIG. 9). The normalcells expressed much less of these Wnt or Fzd proteins, when compared tothe tested HNSCC, with the exception of RPMI 2650. Of note is the lackof detectable Fzd protein in the lysate of the NHBE cell line that had aweakly detectable product by RT-PCR. Beta-catenin was detected in allthe samples, including both HNSCC and NHBE lines.

Example 4 Effects of Anti-Wnt Antibodies and SFRP1

[0089] Treatment with antibody against the extracellular domains ofWnt-1 or Wnt-10b decreased the proliferation of the SNU1076 HNSCC cellline (FIG. 10), while little effect was observed in PCI 13 cells (datanot shown). The inhibition of cell growth by the antibodies wasdependent on the concentration and incubation time. The treatment of theSNU1076 HNSCC cell line with anti-Wnt antibodies, but not controlantibody, also induced apoptosis (FIG. 12). Similar to anti-Wntantibodies, treatment with recombinant SFRP1 protein (2 μg/ml), anatural antagonist of Wnt signaling, inhibited growth of SNU 1076 cells(FIG. 11).

[0090] To determine if the effects of anti-Wnt antibody on SNU1076 cellswere related to inhibition of Wnt signaling, we compared levels of theWnt regulated genes cyclin D1 and fibronectin (FIG. 7A). The anti-Wnt-1antibody, but not the control IgG, reduced cyclin D1, fibronectin, andβ-catenin levels in the cytosol of SNU 1076 cells. To confirm theseresults, TOPFLASH-Luc, a reporter plasmid containing TCF/LEF bindingsites, or FOPFLASH-Luc, a negative control plasmid having mutant bindingsites was introduced into SNU 1076 cells together with the pCMV-β-galplasmid (to assess transfection efficiency). Luciferase activity washigher in the TOPFLASH than the FOPFLASH transfected cells, indicatingthat LEF/TCF dependent transcription was constitutively active. Cellstransfected with FOPFLASH showed no changes in the low baselineluciferase activity after treatment with anti-Wnt1 antibodies, whereascells transfected with TOPFLASH displayed decreased luciferase activity(FIG. 7B).

Example 5 Effects of Anti-Frizzled Antibodies

[0091] Wnt signaling through frizzled receptors has been described toinhibit apoptosis (Chen, S. et al. “Wnt-1 signaling inhibits apoptosisby activating beta-catenin/T cell factor-mediated transcription,” J CellBiol 152:87-96 (2001)). Also some of the genes that are regulated byTCF/beta-catenin are known to be associated with the cell cycle andcellular proliferation. By blocking the binding of Wnt proteins to theirreceptors via antibodies directed to the extracellular portion offrizzled this pathway can be interrupted. Decreasing the downstreamtranslocation of beta-catenin to the nucleus could result in slowertumor growth or death of the cell.

[0092] The immunization strategy that may be useful in terms of raisingspecific antibodies that delay growth in cell culture will then betested for potential in vivo efficacy in mice. Previously we have usedthe H-2^(b) thymoma line EL4 as a syngeneic tumor in C57B1/6 mice (Corr,M. et al., “Costimulation provided by DNA immunization enhancesantitumor immunity,” J Immunol 159:4999-5004 (1997); (Cho, H. J. et al.,“Immunostimulatory DNA-based vaccines induce cytotoxic lymphocyteactivity by a T-helper cell-independent mechanism,” Nat Biotechnol18:509-514 (2000)). This line will be transfected with a human frizzled2 expression vector and selected in neomycin. The expression vector willbe made by excising the frizzled 2 containing insert from one expressionvector with Nde1 and BamH1 and ligating the insert into pcDNA3(Invitrogen) which has a CMV promoter and a neomycin selection cassette.Thirty-two female C57B1/6 mice will be divided into groups of 8 miceeach. They will be injected in the dermis of the tail with a combinationof 50 μg plasmid encoding a costimulator and 50 μg linker plasmiddiluted in normal saline at weeks zero, one and two. A group with emptyvector is included as a negative control. On day 28 the mice will beinjected subcutaneously in the flank with 20×10⁶ frizzled 2 transfectedEL4 cells or untransfected cells (Cho, H. J. et al., “ImmunostimulatoryDNA-based vaccines induce cytotoxic lymphocyte activity by a T-helpercell-independent mechanism,” Nat Biotechnol 18:509-514 (2000)). The micewill be monitored three times a week for weight, and tumor growthmeasured with a caliper. Tumor volume is calculated by lengthxwidth²×π/6as previously described (Radulovic, S. et al., “Inhibition of growth ofHT-29 human colon cancer xenografts in nude mice by treatment withbombesin/gastrin releasing peptide antagonist (RC-3095),” Cancer Res51:6006-6009 (1991)). Mice will be sacrificed four weeks post tumorchallenge or if the tumor burden reaches approximately 2000 mm³.Inhibition of tumor growth will be determined by ANOVA.

[0093] The polyclonal antibodies that are generated by the immunizationstrategies may exhibit binding, but may not be sufficiently concentratedin the polyclonal serum to have a biologic effect. The serum fromseveral immunization strategies may need to be tested in vitro for theirpotential therapeutic utility before proceeding with the in vivo activeimmunization strategy for tumor prevention. The inhibition of tumorgrowth in the murine model may be due to cellular responses as well ashumoral, which will lead to further investigations. These assays may beuseful in determining if the frizzled expressing cell lines aresusceptible to anti-proliferative activity of polyclonal anti-frizzledIgG.

Example 6 Overexpression of Wnt 14 and 16

[0094] Based upon sequences in the public human DNA gene database, weprepared gene-specific primers for all the known human wnt and frizzledgenes. We obtained mRNA from primary human chronic lymphocytic leukemiacells or normal human lymphocytes. Using real time PCR, we then comparedthe relative expression of the wnt and frizzled genes in the normal andmalignant lymphocytes, compared to the control genes GAPDH and 18S mRNA.We discovered that wnt 16 was 70-100 fold overexpressed in the malignantlymphocytes. Wnt 14 was 400 fold overexpressed in the malignantlymphocytes. We sequenced the amplicons to determine their identities.Northern blots of normal human tissues confirmed the lack of significantexpression of wnt 16 mRNA in non-lymphoid cells and in peripheral bloodlymphocytes. Following the procedures described above, we will confirmthe overexpression of wnt 16 and wnt 14 protein in the malignant cellsusing non-crossreactive antibodies, and will test the effects of theanti-wnt 16 and anti-wnt 14 antibodies on cell survival in vitro, usingnormal lymphocytes as a control. In addition, upon review of ourresults, we can develop these antibodies and antigens as therapeuticagents

Example 7 Regulation of Lymphocyte Survival by Integrins

[0095] The survival of lymphocytes requires that they interact with theextracellular matrix proteins produced by stromal cells in theirsurrounding microenvironment. These interactions may render the cellsresistant to spontaneous and drug-induced apoptosis. VLA4integrin-mediated cell adhesion is known to be involved in regulatingcell survival in some leukemic cell lines. We are studying integrineffects on the survival of primary blood lymphocytes. Our data show thatthe α4-CS1 fragment of fibronectin significantly improves the survivalof blood lymphocytes. To develop a potential therapeutic strategy thatcombines integrin antagonists with cytotoxic drugs, we are investigatingthe mechanism of several integrin α4-specific antagonists. Thesecompounds specifically inhibit the adhesion of B chronic lymphocyticleukemia cells to fibronectin. We are currently studying the signalingevents affected by these integrin antagonists in primary humanlymphocytes.

Example 7 Wnt Gene Expression in Normal and Malignant Lymphocytes

[0096] The secreted proteins of the diverse wnt gene family are known toplay an important role in cell growth and differentiation. Evidencesuggests t wnt signaling may regulate apoptosis. The purpose of theseexperiments is to identify the wnt genes that are most highly expressedin resting lymphocytes, and then to determine their potential role incell survival.

[0097] Total RNA was prepared and treated with RNase-free DNase. ThecDNA was synthesized from 5 μg total RNA using Superscript reversetranscriptase and oligo dT. To assure that there was no genomic DNAcontamination, controls in which no reverse transcriptase was added werealso carried out. TaqMan real-time PCR was performed using an ABI PRISM7700 sequence Detector. Primers and probes for 46 wnt family members andtheir related genes were designed using Primer Express version 1.0(Applied Biosystems). The reaction conditions were as follows: 2 min at500 C. (one cycle), 10 min at 950 C. (one cycle), and 15 s at 950 C. and1 min at 600 C. (45 cycles). Two replicates for each gene wereperformed.

[0098] Having developed and validated a TaqMan real-time PCR assay toquantify the gene expression profiles of the wnt family and its relatedgenes, we measured the gene expression profile in three B-CLL, twonormal peripheral blood lymphocyte populations, and one purified B cellsample. We found that wnt6, wnt14 and wnt16 were overexpressed in B-CLL,compared to normal PBL or purified B cells. Wnt14 mRNA levels in B-CLLwere 16-178 times those of PBL and B cell samples. The concentration ofwnt6 mRNA in B-CLL samples was 8-32 fold higher than that in normal PBLand B-CLL samples. Wnt16 mRNA was expressed at 32-178 higher levels inB-CLL than in PBL. For other wnt-related families, such as Fzd, Frp,Wisp and DKK, we did not observe any significant differences. Thus, thewnt gene overexpression appears to be unique.

[0099] We have established a model system to study theintegrin-dependent interaction of primary human lymphocytes withextracellular matrix proteins, and have shown that the binding promotescell survival. We can now test the effects of integrin antagonists oncell signaling and apoptosis in both normal and malignant cells.

[0100] Other experiments revealed three wnt genes that are overexpressedin lymphocytes of patients with B-CLL, compared to normal peripheralblood lymphocytes. Since wnt proteins are secreted, they may function assurvival factors for the malignant cells.

[0101] The specificities of the feeder cell-lymphocyte interactions thatdelay senescence and apoptosis are identified by using purifiedlymphocyte subpopulations (CD4, T cells, CD8, T cells, B cells),co-culturing with different feeder cells (monocytes, dendritic cells,endothelial cells, fibroblasts), and then measuring both spontaneous anddrug-induced apoptosis.

[0102] The specific surface molecules and/or secreted factorsresponsible for the extended survival of the lymphocytes are identifiedby testing the effects of blocking antibodies against surface antigenson the feeder cells and the lymphocytes, determining the effect ofneutralizing antibodies against cytokines and growth factors, andgenerating sense and anti-sense transfectomas of feeder cells to confirmthe roles of the specific interaction revealed in the first two methodsdescribed.

[0103] The intracellular signaling pathways in quiescent lymphocytesthat are altered by contact with feeder cells, and that increase theirsurvival are identified by determining levels and phosphorylation statusof proteins in key activation pathways (mitogen activated proteinkinase, STATs, NF-Kb, b-catenin), assessing levels and phosphorylationstatus of proteins that regulate apoptosis (bc12 family members,caspases, IAPs, SMAC/DIABLO), and testing the effects of pharmacologicinhibitors of signal transduction on the survival of quiescentlymphocytes cultivated with feeder cells, alone or in combination withcytotoxic agents.

[0104] Numerous modifications may be made to the foregoing systemswithout departing from the basic teachings thereof. Although the presentinvention has been described in substantial detail with reference to oneor more specific embodiments, those of skill in the art will recognizethat changes may be made to the embodiments specifically disclosed inthis application, yet these modifications and improvements are withinthe scope and spirit of the invention, as set forth in the claims whichfollow. All publications or patent documents cited in this specificationare incorporated herein by reference as if each such publication ordocument was specifically and individually indicated to be incorporatedherein by reference.

[0105] Citation of the above publications or documents is not intendedas an admission that any of the foregoing is pertinent prior art, nordoes it constitute any admission as to the contents or date of thesepublications or documents.

We claim:
 1. A method for determining overexpression of a wnt orfrizzled gene in a tumor cell comprising: (a) isolating messenger RNAfrom a tumor cell and from a corresponding normal cell from the samesource; (b) performing reverse transcription PCR on the tumor cell andthe normal cell utilizing primers directed against regions of the wnt orfrizzled gene that are non-homologous with a known wnt gene or a knownfrizzled gene and obtaining amplicons from the reverse transcriptionPCR; (c) assessing the concentrations of the resulting amplicons of step(b) in comparison to a control housekeeping gene; and (d) identifyingtumor cells that overexpress a wnt or frizzled gene at about five foldcompared to normal cells tissue from the same source
 2. An isolatedantibody directed against at least one sequence that corresponds to anon-homologous regions of a known wnt gene or a known frizzled gene. 3.A method of detecting overexpression of at least one wnt and/or frizzledprotein in a cancer cell compared to a non-cancer cell comprising: (a)contacting the cancer cell with an antibody directed against at leastone sequence that corresponds to a non-homologous region of a known wntor a known frizzled gene; (b) contacting the non-cancer cell with thesame antibody as in step (a); (c) comparing the interaction of theantibody with the cancer cell to the interaction of the antibody withthe non-cancer cell; and (d) correlating the interactions in (c) withthe expression levels of the wnt and/or frizzled protein in both thecancer cell and the non-cancer cell.
 4. A method for altering the growthof a cell overexpressing at least one wnt and/or frizzled proteincomprising: contacting the cell with a non-crossreactive antibodyagainst the wnt and/or frizzled protein.
 5. The method of claim 4,wherein the antibody is coupled to a cytotoxic agent and/orradioisotope.
 6. A method for altering the growth of a celloverexpressing at least one wnt and/or frizzled protein comprising:contacting the cell with a synthetic peptide, a recombinant protein, ora DNA vector, or any combination of a synthetic peptide and arecombinant protein and a DNA vector, comprising at least onenon-homologous region of known wnt and/or frizzled proteins.